The genome sequence of the blonde ray, Raja brachyura Lafont, 1871

We present a genome assembly from an individual female Raja brachyura (Blonde Ray; Chordata; Chondrichthyes; Rajiformes; Rajidae). The genome sequence spans 2,700.50 megabases. Most of the assembly is scaffolded into 49 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 17.12 kilobases in length. Gene annotation of this assembly on Ensembl identified 24,252 protein-coding genes.


Background
The Blonde Ray (Raja brachyura, Lafont, 1871), is a benthic species of elasmobranch found in coastal waters around the north-east Atlantic.In UK waters it is more commonly found along the west and south coasts and typically shows a preference for sandy bottoms (Serena, 2005).It is light brown with creamy-white patches and numerous dark brown/ black spots which reach the edges of the of the wing and onto the tail (Wheeler & Stebbing, 1978).It can reach sizes of 120cm from nose to tail and weigh up to 18 kg (Stehmann et al., 1984).The Blonde Ray has a varied diet of crustaceans, annelids and fish, with larger, more active crustaceans and fish becoming a more important component of the diet as the fish grows in size (Ellis et al., 1996;Holden & Tucker, 1974).The Blonde Ray mature between ages 4 and 6, and at around 80 cm in length (typically later and larger in females) (Gallagher et al., 2004).As with other Skates, Blond Rays are oviparous typically laying around 30 to 40 eggs in a season (Holden et al., 1971;Porcu et al., 2015).
Raja brachyura is a commercially important species throughout its range (Thys et al., 2022).Its low fecundity, coupled with sexual maturity not being reached until the ray is large and several years old, makes the Blonde Ray vulnerable to fisheries pressure (both targeted and bycatch) (Silva et al., 2012;Thys et al., 2022, Thys et al., 2023).As such it is currently listed as near threatened with a decreasing population trend by the IUCN Redlist (McCully et al., 2015).
Here we present the first chromosomally complete genome for this species and genus.

Genome sequence report
The genome of an adult female Raja brachyura (Figure 1) was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating a total of 24.00 Gb (gigabases) from 1.92 million reads, providing approximately 33-fold coverage.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data, which produced 630.99 Gbp from 4,178.75 million reads, yielding an approximate coverage of 234-fold.Specimen and sequencing information is summarised in Table 1.
Manual assembly curation corrected 119 missing joins or misjoins and one haplotypic duplications, reducing the scaffold number by 5.24%, and increasing the scaffold N50 by 0.57%.The final assembly has a total length of 2,700.50Mb in 1,646 sequence scaffolds with a scaffold N50 of 68.4 Mb (Table 2).The total count of gaps in the scaffolds is 2,269.The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (96.03%) of the assembly sequence was assigned to 49 chromosomal-level scaffolds, representing 48 autosomes and the X sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 3).The X chromosome was identified based on synteny with Carcharodon carcharias (GCA_017639515.1)and Hypanus sabinus (GCA_030144855.1).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Sample acquisition
A Raja brachyura specimen (specimen ID MBA-211013-003A, ToLID sRajBra1) was collected from Whitsand Bay, English Channel, UK (latitude 50.33, longitude -4.24) on 13th October 2021.The specimen was taken from its habitat of shell and sand using an otter trawl deployed from RV Sepia.The specimen was identified by Rachel Brittain (Marine Biological Association) based on gross morphology.The fish was first anaesthetised and then overdosed using Aquased (2-phenoxyethanol).Destruction of the brain was used as a secondary method to ensure the animal was deceased before tissue sampling took place as in accordance with Schedule 1 methodology under the home office licence.Samples taken from the animal were preserved on dry ice.
The initial identification was verified by an additional DNA barcoding process according to the framework developed by Twyford et al. (2024).A small sample was dissected from the specimen and stored in ethanol, while the remaining parts of the specimen were shipped on dry ice to the Wellcome Sanger Institute (WSI).The tissue was lysed, the COI marker region was amplified by PCR, and amplicons were sequenced and compared to the BOLD database, confirming the species identification (Crowley et al., 2023).Following whole genome sequence generation, the relevant DNA barcode region was also used alongside the initial barcoding data for sample tracking at the WSI (Twyford et al., 2024).The standard operating procedures for Darwin Tree of Life barcoding have been deposited on protocols.io(Beasley et al., 2023).

Nucleic acid extraction
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.In sample preparation, the sRajBra1 sample was weighed and dissected on dry ice (Jay et al., 2023).Gill tissue was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).HMW DNA was extracted using the Nanobind Blood extraction protocol (Denton et al., 2023b).DNA was sheared into an average  also generated from gill tissue of sRajBra1 using the Arima-HiC v2 kit.The Hi-C sequencing was performed using pairedend sequencing with a read length of 150 bp on the Illumina NovaSeq 6000 instrument.
The mitochondrial genome was assembled using MitoHiFi (Uliano- Silva et al., 2023), which runs MitoFinder (Allio et al., 2020) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Assembly curation
The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants (ASCC) pipeline (article in preparation).Flat files and maps used in curation were generated in TreeVal (Pointon et al., 2023)  and removed.Sex chromosomes were identified by synteny.The entire process is documented at https://gitlab.com/wtsi-grit/rapid-curation (article in preparation).

Evaluation of the final assembly
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020) et al., 2020) andBUSCO scores (Manni et al., 2021;Simão et al., 2015) were calculated.
The genome assembly and evaluation pipelines were developed using the nf-core tooling (Ewels et al., 2020), use MultiQC (Ewels et al., 2016), and make extensive use of the Conda package manager, the Bioconda initiative (Grüning et al., 2018)

Genome annotation
The Ensembl Genebuild annotation system (Aken et al., 2016) was used to generate annotation for the Raja brachyura assembly (GCA_963514005.1) in Ensembl Rapid Release at the EBI.Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: The authors have submitted a good quality assembly of the blond ray, Raja brachyura.The assembly was done with PacBio HiFi and HiC using the Hifiasm assembler which is a standard approach at this point for good draft assemblies.I do not have any major concerns with the approach or results.It would be interesting to see a comparison between the primary haplotype and the secondary contigs.What is the snv rate between the two chromosomes?How much genetic diversity is stored in the shrinking populations of blond rays?These are not essential points, but are potentially of interest.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Stephan Koblmüller
University of Graz, Graz, Austria Adkins et al. present the genome assembly of the blonde ray, Raja brachyura.The data are clear and the approaches are well described and I have no objections to any of the data presented.As far as I can tell, the datasets are deposited in publicly accessible repositories.
The background section gives a concise, but nonetheless pretty comprehensive introduction to the study species.Though it is clear to me that providing a high-quality reference genome for this species will serve as basis for numerous other studies, the rationale of why actually sequencing a high-quality reference genome for this species is missing in the background section.In my opinion, one to two sentences on the rationale are required.
In addition, I think, one sentence needs to be rephrased: The Blonde Ray mature between ages 4 and 6, and at around 80 cm in length (typically later and larger in females) (Gallagher et al. 2004) Here "mature" should be replaced by "matures" and I guess with "between ages 4 and 6" you mean between 4 and 6 years, right?I suggest you rephrase this so that it's clear for the reader what you mean.

Is the rationale for creating the dataset(s) clearly described? Partly
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: phylogenetics-/genomics, population genetics/genomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.Reviewer Expertise: Genetics and biotechnology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 1 .
Figure 1.Photograph of the Raja brachyura (sRajBra1) specimen used for genome sequencing: A. Dorsal view, B. Ventral view.

Figure 2 .
Figure 2. Genome assembly of Raja brachyura, sRajBra1.1:metrics.The BlobToolKit snail plot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 2,700,467,673 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (187,439,314 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths(68,366,687 and 26,303,439 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the vertebrata_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Raja%20brachyura/dataset/CAUPSL01/snail.

Figure 3 .
Figure 3. Genome assembly of Raja brachyura, sRajBra1.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Raja%20brachyura/dataset/CAUPSL01/blob.

Figure 4 .
Figure 4. Genome assembly of Raja brachyura sRajBra1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Raja%20brachyura/dataset/CAUPSL01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Raja brachyura sRajBra1.1:Hi-C contact map of the sRajBra1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https:// genome-note-higlass.tol.sanger.ac.uk/l/?d=DS9CH9xQRVC81LrwHEfXaQ.

Reviewer Report 28 ○
August 2024 https://doi.org/10.21956/wellcomeopenres.25132.r93550© 2024 Senevirathna J.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Jayan D.M SenevirathnaUva Wellassa University, Badulla, Uva Province, Sri Lanka I accept this article for indexing in Open research.However, the followings are suggested to improve the article:In keywords; Raja brachyura should be italic.○Inbackground; north-east Atlantic > north-east Atlantic Ocean.○InGenome annotation report; Recheck the number of non-coding genes in the text and the given link.○Inmethods; For HMW DNA extraction, why did you specifically select the gill tissue instead of a muscle...?Is the rationale for creating the dataset(s) clearly described?YesAre the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesCompeting Interests: No competing interests were disclosed.

Table 2 . Genome assembly data for Raja brachyura, sRajBra1.1. Genome assembly
Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Rhie et al. (2021)benchmarks are adapted from column VGP-2020 of "Table1: Proposed standards and metrics for defining genome assembly quality" fromRhie et al. (2021).**BUSCOscoresbased on the vertebrata_odb10 BUSCO set using version 5.4.3.C = complete [S = single copy, D = duplicated], F = fragmented, M = missing, n = number of orthologues in comparison.A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/Raja%20brachyura/dataset/CAUPSL01/busco.RNA was extracted from gill tissue of sRajBra1 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol(do Amaral et al.,  2023).The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit.ing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences Sequel IIe (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were

Table 3 . Chromosomal pseudomolecules in the genome assembly of Raja brachyura, sRajBra1. INSDC accession Name Length (Mb) GC%
Table 4 contains a list of relevant software tool versions and sources.

Table 4 . Software tools: versions and sources. Software tool Version PRJEB61690
(Wellcome Sanger Institute, 2023).The genome sequence is released openly for reuse.The Raja brachyura genome sequencing initiative is part of the Darwin Tree of Life (DToL) project.All raw sequence data and the assembly have been deposited in INSDC databases.Raw data and assembly accession identifiers are reported in Table1 and Table 2.